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WORLD INTELLECTUAL PROPERTY ORGANIZATION
Internationa] Bureau

PCT

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(51) International Patent Classification 6 :
G01N 21/64, 15/14

(11) International Publication Number: WO 99/60383

(43) International Publication Date: 25 November 1999 (25. 11 .99)

(21) International Application Number: PCT/US997 10874

(22) International Filing Date: 17 May 1999 (17.05.99)

(30) Priority Data:

6XV085,545

15 May 1998 (15.05.98)

(71) Applicant (for all designated States except US): FLUORRX,

INC. [US/US]; 979 Keystone Way, Carmel, IN 46032 (US).

(72) Inventor; and

(75) Investor/Applicant (for US only): SZMACINSKI, Henryk
[P1VUS]; 979 Keystone Way, Carmel, IN 46032 (US).

(74) Agent: KENEMORE, Max; FluorRx, Inc., 979 Keystone Way,
Carmel, IN 46032 (US).

(81) Designated States: CA, JP, US, European patent (AT, BE, CH,
CY, DB, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL,
PT, SE).

Published

With international search report

(54) Title: IMPROVED PHASE ANGLE AND MODULATION OF FLUORESCENT ASSAY
(57) Abstract

The presence or concentration of analytes in a sample can be assayed by observing with fluorescent lifetime techniques such as
phase angle shift and modulation change the combined response to excitation of a fluorescent probe in the presence of the sample and a
fluorescent material, the probe having a fluorescence intensity response to the presence or concentration of the analyte, the probe and the
material having no substantial fluorescence lifetime change in response to the presence or concentration of the analyte and the material
having a fluorescence lifetime m response to excitation that is different from the fluorescence lifetime of the probe.

FOR THE PURPOSES OF INFORMATION ONLY

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WO 99/60383

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Title

IMPROVED PHASE ANGLE AND MODULATION OF FLUORESCENT ASSAY

WO 99/60383

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Description

5 At present, there is considerable interest and

research activity in the field of chemical sensing.
Rapid and continuous monitoring of many analytes { pH,
pC0 2 , 0 2 , metal ions, etc.) Is required in many areas of
science, including analytical chemistry, biochemistry,

10 environmental sensing, clinical chemistry and industrial
applications. Fluorescence-based sensing is one of the
promising techniques because of fluorescence
sensitivity, providing number of sensitive and specific
fluorescent probes for a variety of analytes and their

15 fabrication with fiber optics.

At present, most types of commercial fluorescence
sensing devices are .based on the standard intensity-
* based methods, in which the intensity of the
fluorescence produced by the probe molecule changes in

20 response to the analyte of interest. These intensity
changes can be induced by an analyte due to changes in
extinction coefficient, changes in quantum yield,
absorption and emission spectral shifts, or simply due
to the inner filter effects. While intensity

25 measurements are simple and accurate in the laboratory,
they are often inadequate in real-world situations. This
is because the sample may be turbid, the optical
surfaces may be imprecise and become dirty and optical
alignment may vary from sample to sample. A significant

30 disadvantage of intensity based sensing is the problem
of referencing the intensity measurements. The

WO 99/60383 PCT/US99/10874

fluorescence intensity measurement depends on the
intensity of exciting light, the optical density at the
excitation and emission wavelengths, the light loses in
the optical path length, detector sensitivity and the

5 concentration of the fluorophore. These difficulties
with intensity-based sensing appear to be limiting the
more widespread use of fluorescence for quantitative
chemical sensing.

Recent advances in optoelectronic have now made

10 possible a new type of fluorescence sensing. Instead of
fluorescence intensities it is possible to measure
fluorescence lifetime, particularly by the phase
modulation method with a simple light sources. The
advantages of lifetime-based sensing and the for a

15 several mechanisms of analyte-induced changes in
lifetime are reviewed elsewhere in detail (Szmacinski
and Lakowicz, Topics in Fluorescence Spectroscopy, Vol
4, pp. 295-334, Plenum Press, New York 1994) . The
preferred lifetime-based sensing technique is phase-

20 modulation, where analyte-induced changes in lifetime of
the probe are measured by phase angle and modulation at
single modulation frequency. The phase and modulation
are related to the analyte concentration. A number of
lifetime-sensitive probes have been characterized for

25 several analytes such as pH, Ca 2+ , Mg 2 *, K*, Na + .
Practically all of the known analyte lifetime-sensitive
probes excluding the probes for 0 2 sensing display short
lifetimes, most often in the range of 1- 5 ns. Using
probes with short lifetimes requires high modulation

30 frequencies in the range of 50 - 300 MHz in order to
obtain sufficient changes in phase and modulation for

WO 99/60383 PCT/US99/ 10874

analyte sensing. However, even though inexpensive light
sources such as LED' s can be modulated in that range of
frequencies, the cost of phase modulation device is
. still sufficiently expensive to inhibit broad commercial
5 use.

There is observed a significant effort in several
laboratories to develop functional long lifetime probes
having a fluorescence lifetime in the range above 100
ns. There is a large number of fluorophores that display

10 long lifetime fluorescence, such as metal-ligand

complexes based on ruthenium, rhenium, osmium, platinum
or rhodium. Lifetimes as long as 100 yis can be obtained
using such probes . However, there are not known such
metal-ligand complex-based probes that are sufficiently

15 sensitive to analytes for practical use. Only 0 2 probes
based on metal-ligand complexes in which the mechanism
of quenching is exploited are presently used widely.
The advantage of longer lifetime probes is that sensing
can be at a relatively lower modulation frequency, for

20 example, in '.the range . of 10-1000 kHz. A phase and/or
modulation instrument based on the use of longer
lifetime fluorescent materials, such as the metal-ligand
complex based materials can be designed to use
inexpensive components due mainly to the lower

25 frequencies required.

It is among the objects of the present invention to
.;■ overcome the disadvantages of the present technology.

It is one object to avoid the need for development
and manufacture of relatively exotic long lifetime

30, fluorescent materials such as those based on metal-
ligand complexes.

WO 99/60383 PCT/US99/1 0874

It is another object of this invention to avoid the
need for the use of high frequencies in phase-modulation
assays, so that relatively less expensive lower
frequency electronic components can be used.
5 It is yet another object of this invention is to

measure phase and modulation in the low modulation
frequencies using available (or designed) fluorophores
in which the intensity is sensitive to the analyte of
interest .

10 It is a further object of this invention to avoid

the problems encountered in the past with intensity-
based fluorescent assays due to ambient light, turbidity
in the sample, the need for optical couplings, intensity
dissipated in wave guides, and the like.

15 These and other objects are accomplished by the

present invention, which makes use of the discovery that
relatively low frequency phase and modulation techniques
can be used to assay for analytes by employing, in
combination, a fluorescent probe that shows a

20 fluorescent intensity response in the presence of the
analyte but has a fluorescent lifetime that is
substantially unaffected by the presence of the analyte
and a fluorescent material having a fluorescent lifetinie
that is different from the fluorescent lifetime of the

25 probe, the fluorescent material being substantially
unaffected in both fluorescence lifetime and
fluorescence intensity by the presence of the analyte.

Although the mechanism by which this invention
works is not fully understood at the present time, it is

30 believed' that the phase angle and modulation of the
sample depend on values of lifetime and fractional

•5

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intensities of components . The changes in phase angle
and modulation can be as result of changes in fractional
intensities without changes in the lifetime of both
component. By mixing an analyte sensitive fluorophore
5 (short lifetime or long lifetime) with the second
fluorophore that is not analyte sensitive (long lifetime
or short lifetime) and by correctly selecting the
relative concentration of the f luorophores, the
excitation wavelength, and the emission band observed,

10 an analyte sensitive probe can be created. The expected
analyte induced changes in phase angle and modulation
can be as large as 90 degree and 1.0, respectively. To
observe large changes in phase angle and modulation the
modulation frequency can be at a lower range, determined

15 by the long lifetime component.

The controlled mixing of two fluorophores allows
using any intensity-based fluorophore regardless of its
lifetime as a lifetime-based probe using a phase and
modulation technique. The analyte-induced changes in

20 fractional intensities of two components allow the
determination of the, analyte concentration from the
phase and/or modulation at a single modulation
frequency.

The properties, requirements, ri . advantages and
25 various applications of the present- invention are
discussed below with reference to the drawings.-

Brief Description of the Drawings

Figs, la and Fig. lb show the expected frequency
responses of phase angle and modulation for long

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lifetime and short lifetime f luorophores .

Fig. 2a and Fig 2b show the expected frequency
responses of phase angle and modulation of fluorescence
that consist of fraction of long lifetime and fraction
5 the short lifetime fluorescence.

Fig. 3a and Fig. 3b show the expected frequency
responses of phase angle and modulation where the value
of short lifetime is changed from 0.5 to 10 ns in
several steps.

10 Fig. 4a and Fig 4b show the expected frequency

responses of phase angle and modulation where the value
of long lifetime is 100, 500, and 5000 ns and short
lifetime fluorescence of 10 ns.

Figs. 5a, 5b, 6 and 7 illustrate Example 1.
15 Figs. 8, 9 and 10 illustrate Example 2.

Figs. 11, 12, 13, 14 and 15 illustrate Example 3.

Detailed Description

Referring again to Figs, la and lb, two distinct
20 ranges of modulation frequencies are needed to measure
the short lifetime and to measure long lifetime
fluorescence. In order to measure the lifetimes shorter
than 5 ns that display most of organic f luorophores,
high modulation frequencies are required in order of 100
25 MHz, This is normally achieved by an expensive phase-
modulation "fiuorometers which are commonly' used in
research labs. Long lifetime fluorescence requires low
modulation frequencies in the range of 100 kHz. The
design of a phase-modulation instrument for low
30 modulation- frequencies is less expensive and can provide
higher accuracy of measurements that similar devices

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with high frequency. However, there are not presently
widely available lifetime-sensitive fluorescence sensors
that display long lifetimes other than those employing
metal-ligand complexes and used in oxygen sensing as
5 described above.

Figs. 2a and Fig 2b show the expected frequency
responses of phase angle and modulation of fluorescence
that consist of a fraction of long lifetime and a
fraction the short lifetime fluorescence. The values

10 from 0 to 1 represent the fractional intensity of short
lifetime fluorescence in the measured signal. There are
observed great changes from 0 to about 90 degrees in
phase angle and from 1 to 0 in modulation values upon
changes of fractional contribution of fluorescence from

15 both fluorophores. In addition the steeples part of the
modulation value is equal the fractional intensity of
short lifetime fluorescence. Thus fractional-dependent
phase angle and/or modulation can be used to measure the
intensity of a desired f luorophore in the sample using

20 in most cases only one modulation frequency. The changes
in fractional intensity can be induced by the analyte;
(1) by affecting the absorption spectra (extinction
coefficient and/or spectra shift), (2) by affecting the
emission spectra (quantum yields and/or spectra shifts).

25 There are many possibilities to optimize such a sensor
probe by the choice of excitation wavelength/ emission
band and relative concentration of used fluorophores.

Referring again to Figs. 3a and 3b, the fractional
intensity of the short fluorescence lifetime component

30 in each case is the same for 0.15 in Fig. 3a and 0.5 in
Fig. 3b. The important observations from these figures

WO 99/60383 PCT/US99/1 0874

are that the phase angle and modulation below certain
frequency are not sensitive to the value of short
lifetime fluorescence in the sample. This is similar to
the gating technique in the pulse method where, by

5 applying a certain delay after pulse excitation, only
the signal from long lifetime fluorescence is detected.
In the phase-modulation technique it is impossible to
measure only long the lifetime component. Analytical
methods have been developed for background correction in

10 phase-modulation f luorometry based on the measurements
of the background sample or based on known intensity
decay of background and it contribution in the sample
signal. In the case presented in Fig. 3a and 3b the
desired intensity of the long lifetime component can be

15 obtained by measuring the phase and/or modulation at
single modulation frequency regardless of intensity
decay of the background or autofluorescence or scattered
light until the mean lifetime is short enough compared
to a long lifetime fluorophore. This feature can be used

20 also to determine the anisotropy of the long lifetime
component in turbid media with scattered light or with
background fluorescence having a known value of
anisotropy. This may find immediate application in
detecting binding of high molecular weight

25 macromolecules labeled with a metal-ligand complexes or
in immunoassays. It is also important for- chemical
sensing that changes in the lifetime due to presence of
an analyte for short lifetime indicators have no effect
of fractional intensity and thus on sensing of analyte

30 concentration.

Referring again to Figs 4 a and 4b, the most

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important observation is from Fig 4b where the value of
steeples part of modulation reflect the fractional
intensity of the short lifetime component regardless on
the value of the long lifetime component. Also, it is
important that the choice of low modulation frequency
depends mostly on the value of the long lifetime
component but not on the value of short lifetime '
component.

Examples

The present invention is further illustrated with
reference to the following examples. Example 1
demonstrates the phase and modulation sensitivity when
the fractional intensity of sample is varied by various
relative concentrations of two dyes in the sample.

Example 2 demonstrates the possibility to determine
the intensity of flurophore of interest in presence of
various araoun of background or autofluorescence from the
solvent.

Example 3 demonstrate how the sensing probe can be
created when pH induced changes in fractional
intensities of , a probe contained pH intensity sensitive
indicator and long lifetime f luorophore con be measured
by phase angle and modulation.

Example 1 . .

Two fluorophores have been chosen, one with a long
lifetime fluorescence from met al-ligand complexes like
[Ru(bpy) 2 dcbpyj? with a lifetime in glycerol of 1060 ns
and the second with short lifetime like many organic
fluorophores Texas Red Hydrazide with a lifetime of 3.4

WO 99/60383 PCT/US99/1 0874

hs in glycerol. The two dyes were mixed at various
relative concentrations to induce the various fractional
intensities in the sample

Fig/ 5a show the absorption spectra of long

5 lifetime fluorophore [Ru(bpy) 2 dcbpy] 2+ and short lifetime
fluorophore Texas Red Hydrazide (TRH) (solid lines and
their mixture at concentrations specified in Figure. It
is shown that any excitation wavelength shorter than
about 640 nm will excite both f luorophores . The

10 resulting fractional intensities from both fluorophores
will be strongly dependent on the choice of excitation
wavelength. One can imagine that value of extinction
coefficient or shift in absorption spectrum wiir result
in changes of fractional intensities that can be

15 monitored with phase and/or modulation measurements. One
excitation wavelength has been chosen as 488 nm (Argon-
ion laser) . The total concentration of dyes were low to
avoid the inner filter effects. The changes in
absorption was induced by using various concentration

20 combination of both fluorophores.

Fig. 5b shows the emission spectra of
[Ru(bpy) 2 dcbpy] 2+ and TRH at one selected concentration
combination. The emission spectra overlap well and for
phase and modulation measurements we used the long pass

25 filter above 550 nm.

Fig. 6 show the frequency responses of phaie angle
for long lifetime fluorophore [Ru(bpy) 2 dcbpy] 2+ with a
lifetime of 1060 ns and the short lifetime TRH of 3.4 ns
when mixed together at a specified relative

30 concentrations from 0 to 12.8. The obtained values for
fractional intensities are in good agreement with those

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expected from steady-state measurements of full emission
spectra. These experimental data are related to those
simulated and discussed in Pig. 2a. r

Fig. 7 show the frequency responses of modulation
5 for long lifetime fluorophore [Ru (bpy) 2 dcbpy] 2+ with a
lifetime of 1060 ns and the short lifetime TRH of 3.4 ns
when mixed together at a specified relative
concentrations from 0 to 12.8. These experimental data
confirm that presented and discussed in Fig. 2b.

Example 2

The purpose of this example was to demonstrate the
calculation of intensity of long lifetime fluorophore in
presence of background fluorescence from the solvent.

15 Long lifetime fluorophore was the same as in Examplel
[Ru(bpy) 2 dcbpy] 2 with a lifetime in glycerol of 1060 ns.
The glycerol (from Fluka) displayed a background
fluorescence that overlaps with the emission .- of
ruthenium. In many applications the requirements are for

20 very low dye concentration which posses the difficulties
for increased background corrections. The increased
contribution of background fluorescence from solvent was
obtained by the dilutions of the ruthenium sample with
glycerol.

25 Fig. 8 show the emission spectra of ruthenium with

decreased^ concentrations and also background
fluorescence from used glycerol. The fractional
intensity of glycerol calculated by integrating the
spectra are following: 0.108, 0.379, 0,757, and 0.886 at

30 ruthenium concentration of 740, 150, 29 and 6' nM.

Fig. 9 show frequency responses of phase angle of

WO 99/60383 PCT/US99/1 0874

the samples with increased contributions of background
fluorescence. The obtained values are in good agreement
with those from steady-state measurements . The small
difference are because of different excitation sources

5 (xenon lamp and monochromator in steady-state, and Ar-
ion laser in phase-modulation measurements) . It should
be noted that phase angle is related only to fractional
intensity at modulation frequencies lower than 1 MHz.
The glycerol displayed a complex intensity decay with a

10 mean lifetime shorter than 3.5 ns. These experimental
data confirm that presented in Fig 3a where the short
lifetime component do not contribute to changes in phase
angle for certain low modulation frequencies .

Fig 10 show frequency responses of modulation of

15 the samples with increased contributions of background
fluorescence. The steeples part of modulation indicate
good separation between the fluorescence of ruthenium
and that of glycerol, and can be easy used to determine
the absolute intensity of the ruthenium. These results

20 confirm that discussed in Fig. 3b.

Example 3

•' The goal of this example is to demonstrate the
great opportunity of designing the fluorescence probe

25 for measuring a large variety of chemical species where
the change in fluorescence intensity can be 'obtained.
For example we have chosen a pH intensity sensitive
indicator Naphtofluorescein and the second dye with a
long lifetime [Ru(phn) 3 J 2+ . The Naphtofluorescein as most

30 of fluorescein dyes display pH sensitive absorption
spectrum and decreased fluorescence quantum yield at

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lower values of pH. To demonstrate the practical use of
such sensor we used inexpensive blue LED as a excitation
source.

Fig. 11 shows the emission spectra of a mixture of
5 ruthenium and Naphtof luorescein at various values of pH.
The increased pH values affect the fractional
intensities from both of dyes which is displayed as
decreased fluorescence from the ruthenium and increased
contribution from the Naphtof luorescein. The fractional

10 intensities in the sample can be selected by the cutt
off filter or by band pass filter. We have chosen use
long pass filter above the 595 nm. The excitation source
was a blue LED with a maximum intensity at 475 nm.

Fig. 12 shows the frequency responses of phase

15 angle of such pH sensor. There are observed remarkably
large changes in phase angle at modulation induced by
the pH of a sample. The pH phase-based sensing can be
performed at low modulation frequencies in spite of very
short lifetime of Naphtof luorescein of about 0.45 ns

20 frequencies below 10 MHz .

Fig. 13 shows large changes in modulation induced
by pH of the sample. There is a wide range of modulation
frequencies where modulation value is related only to
the pH value even not to modulation frequency. This is

25 because the difference in lifetimes of ruthenium and
Naphtof luorescein is very large about 1000-fo~ld. It is
again important to note that long lifetime value
determines the useful low modulation frequency for
sensing.

30 Fig. 14 shows pH-dependent phase angle for several

modulation frequencies. It should be noted the magnitude

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of phase angle changes up to 69 deg (see values in the
brackets) . This is remarkably pH sensor, which allows
measurements the pH changes as small as of 0.0035 of pH
. unit assuming that phase angle can be measured with an
5 accuracy of 0.1 deg (from curve at 2200 kHz in the range
from pH 6 to 8) . Also choosing the modulation frequency
allow to shift the apparent pKa, in presented case from
6.41 to 7.24.

Fig. 15 shows pH-dependent modulations for several
10 modulation frequencies. The pH induced changes in
modulation ( values in the brackets) are large and
significantly depends on the choice of modulation
frequency. The apparent pKa is slightly dependent on
modulation frequency .
15 The above description is intended to be

illustrative of the present invention, which is intended
to be limited only by the claims that follow.

20 :

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Claims

10 What is claimed is:

1. A method for performing an assay for the presence
or concentration of an analyte in a sample, the,,
15 method comprising:

a, contacting the sample with a fluorescent probe
that, when in the presence of the analyte and
illuminated with activating radiation, emits

20 fluorescent radiation at an intensity that is

related to the presence or concentration of
the analyte, the fluorescent radiation having
a first lifetime that is substantially
unchanged by the presence or concentration of

25 the analyte in the sample;

b. illuminating with modulated activating
radiation of one or more frequencies or
30 amplitudes, or both, the fluorescent probe and

a fluorescent material that emits, in response

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to said activating radiation, fluorescent
radiation having a second fluorescent lifetime
that is substantially unchanged by the
presence or concentration of the analyte and
that has an intensity that is substantially
unchanged by the presence or concentration of
the analyte and wherein the second fluorescent
lifetime is longer or shorter than the first
fluorescent lifetime;

c. sensing change in phase angle or modulation,
or both, of the mixed emissions from the
fluorescent probe and fluorescent material
15 upon such contacting; and

d. determining from such change the presence or
concentration of the analyte.

The method of Claim 1 wherein the sample is
contacted by the fluorescent material.

25 3. The method of Claim 1 wherein the sample is

contacted with a matrix material that supports the
fluorescent probe and, optionally, the fluorescent
material.

The method of Claim 1 wherein the sample , is mixed

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with the fluorescent probe and, optionally, with
the fluorescent material.

An assay device for use in determining the presence
or concentration of an analyte in a sample, the
device comprising

a. a fluorescent probe that, when in the presence of
the analyte and illuminated with activating
radiation, emits fluorescent radiation at an
intensity that is related to the presence or
concentration of the analyte, the fluorescent
radiation having a first lifetime that is
substantially unchanged by the presence or
concentration of the analyte in the sajnple;

b. a fluorescent material that emits, in response to
said activating radiation, fluorescent radiation
having a second fluorescent lifetime that is
substantially unchanged by the presence or
concentration of the analyte and that has an
intensity that is substantially unchanged by the
presence or concentration of the analyte and
wherein the second fluorescent lifetime is longer
or shorter than the first fluorescent lifetime ;
and

c. a support member adapted for supporting the probe
and, optionally, the material in contact with the
sample during illumination of the probe and the

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10
15
20
25

material by the activating radiation.

The device of Claim 5 wherein the support member
comprises a polymeric matrix.

An assay composition for use in assaying for the
presence or concentration of an analyte in a
sample, the composition comprising:

a. a fluorescent probe that, when in the presence
of the analyte and illuminated with activating
radiation, emits fluorescent radiation at an
intensity that is related to the presence or
concentration of the analyte, the fluorescent
radiation having a first lifetime that is
substantially unchanged by the priesence or
concentration of the analyte in the sample; and

b. a fluorescent material that emits, in response
to said activating radiation, fluorescent
radiation having a second fluorescent lifetime
that is substantially unchanged by the presence
or concentration of the analyte and that has an
intensity that is substantially unchanged by the
presence or concentration of the analyte and
wherein the second fluorescent lifetime is
longer or shorter than the first fluorescent

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lifetime,

8. A kit for use in performing an assay for the

presence or concentration of an analyte, the kit
5 comprising:

a. a container containing a fluorescent probe

that, when in the presence of the analyte and
illuminated with activating radiation, emits

10 fluorescent radiation at an intensity that is

related to the presence or concentration of
the analyte, the fluorescent radiation having
a first lifetime that is substantially
unchanged by the presence or concentration of

15 the analyte in the sample; and

b. a container containing a fluorescent material
that emits, iii response to' said activating
radiation, fluorescent radiation having a
second fluorescent lifetime that is
substantially unchanged by the presence or
concentration of the analyte and that has an
intensity that is substantially unchanged by
the presence or, concentration of the analyte
and wherein the second fluorescent litetime is
\ longer or shorter than the first fluorescent
lifetime ♦

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Modulation Frequency (MHz)

Fig. 1a

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1E-3 0.01 0.1 1 10 100 1000

Frequency ( MHz)

Fig. 1b

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0.1 1 10 100

Modulation Frequency (MHz)

Fig. 2a

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4/20

0.1 1 10 100

Frequency (MHz)

Fig. 2b

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5/20

Fig. 3a

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6/20

1.0

Modulation Frequency (MHz)

Fig. 3b

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Modulation Frequency (MHz)

Fig. 4a

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8/20

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9/20

400 450 500 550 600 650

Wavelength (nm)
Serie B ct,* o.ai tvh* o.oozl, (ifes*^

Dyes were in glycerol (Fluka).

Concentration calculated base on extinction coefficients :

Ru- 12,000 Nfcm' 1 , [Evald]

TRH- 80,000 Nfcrn* 1 [MP]

Set,..-

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8
7
6
5
4
3
2
1
0

Ru + TRH (750 + 375 nM)
Exc 488 nm, in glycerol, t=22 C

TRH (375 nM)

Ru(750nM)

550

600 650 700

Wavelength (nm)

750

Fractional intensity in the mixture (serie C):
glyc. - 0.065

Ru - 0.439? os>-o.^oifZ j

TRH - 0.613 J <*1 **:°oW :M lAI" ] ,

1 ob o.si* I r

If separately it t is about 11% more than in mixture.

5 h

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0.01 0.1 1 10 100

Frequency (MHz)

Fig. 6

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Modulation Frequency (MHz)

Fig. 7

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13/20

PCT/US99/10874

Fig. 8

WO 99/60383 PCT/US99/10874

14/20

Frequency (MHz)

Fig. 9

WO 99/60383

15/20

PCT/US99/10874

Modulation Frequency (MHz)

Fig. 10

WO 99/60383

PCT/US99/10874

16/20

160
140
120

[Rufphny 2 *
Long lifetime dye
Mixtures ✓ " " n

( )

Naphtofluorescein

/ \ pH indicator

> \
/ \
/ \
/ \

/ pH6.0 \ I

550 600 650 700 750
Emission Wavelength (nm)

Fig. 11

WO 99/60383

17/20

PCT/US99/10874

WO 99/60383

18/20

PCT/US99/10874

Modulation Frequency (MHz)

Fig. 13

WO 99/60383

19/20

PCT/US99/10874

Fig. 14

WO 99/60383

20/20

PCT/US99/10874

1.0

pH
Fig. 15

INTERNATIONAL SEARCH REPORT

International application No.
PCT/US99/10874

. CLASSIFICATION OF SUBJECT MATTER
IPC(6) : GOIN 21/64, 15/14
US CL :Please Sec Extra Shed.
According to International Patent Classification (IPC) or to both national classification and IPC

FIELDS SEARCHED

Minimum documentation searched (classification system followed by classification symbols)
U.S. : 250/461.2, 458.1, 461.1, 459.1, 461.2, 302; 436/172, 527, 521, 169, 501

Documentation searched other than minimum documentation to the extent that such documents arc included in the fields searched
NONE

Electronic data base consulted during the international search (name of data base and, where practicable, search terms used)
CAPLUS, WPIDS, USPAT, DIALOG, MEDLINE

FLUORESC, PHASE, MODULATE, FREQUENT, PROBE, AMPLITUDE, LIFETIME, FLUORMET, POLYMERIC
MATRI. : _

DOCUMENTS CONSIDERED TO BE RELEVANT

Category*

Citation of document, with indication, where appropriate, of the relevant passages

Relevant to claim No.

WO 92/07245 Al (THE UNIVERSITY OF MARYLAND) 30 April
1992, pages 3-43.

WO 92/13265 Al (THE UNIVERSITY OF MARYLAND) 06
August 1992, whole document, especially pages 9-48.

US 5,270,548 A (STEINKAMP) 14 December 1993, whole
document especially columns 3-10.

US 5,315,122 A (PINSKY et al) 24 May 1994, column 2, lines 59-
68.

1-8

1-7

| | Further documents are listed in the continuation of Box C. Q See patent family annex.

•L"

•o*

Special cate»,ooae of citad <Jooaa«itr

document dafknne, the general atete of the art which »
to be of particular reliance

r document published on or after the intrmstion

tkfa mwt throw doubtt on priority craon(s) or which a
cited to eetabfoh the publication date of another citation or <rfbar

I (as apacifiad) 1

tpubnebeda

tpub&bed prior to tha

(date >XT

filinf date hot later than

tetardoo r

_ i hut chad to

tha priocipte or theory uadartyin$ "th* iirraotioo

documant of particular relevance; tha clainad arrantion
considered novel or cannot ba coosidared to mrorre as
whan tha document ia tekao alone

documant of particular ralaranca; tha claimed invention «
cooaadared to inroKa an rarentrra atep whan the doc
combined with one or nor* other eucfa document*, tnch cot
beans obvioua to a parson skilled in the art

docuaeeut mexabar of tha asm a patent faraUy

the

t b

Date of the actual completion of the international search
04 AUGUST 1999

Name and mailing address of the ISA/US
Corammwocr of Patenta and Trademarks

Box per

Washington, DC 20231
Facsimile No. (703) 305-3230

Date of m ailing of the international search report

30AUG1959

Authorized officer

PADMA BASKAR
Telephone No. (703)

308-0196 Vj

Form PC17ISA/210 (second. sheet)(July 1992)*

INTERNATIONAL SEARCH REPORT

International application No.
PCT/US99/10874

A. CLASSIFICATION OF SUBJECT MATTER:
USCL :

250/461.2, 458.1, 461.1, 459.1, 461.2, 302; 436/172, 527, 521, 169, 501

Form PCT/1SA/210 (extra sheet)(July 1992)*

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